M. tuberculosis RNA polymerase alpha subunit

ABSTRACT

The present invention provides isolated nucleic acids encoding RNA polymerase alpha subunit from M. tuberculosis, vectors comprising the nucleic acids, cells comprising the vectors, and methods for producing M. tuberculosis alpha subunit. The invention also provides in vitro and in vivo methods for high-throughput screening to identify inhibitors of M. tuberculosis RNA polymerase.

This application is a 371 of PCT/US97/22216, filed Dec. 3, 1997, which claims the benefit of Ser. No. 60/032,292 filed Dec. 3, 1996.

FIELD OF THE INVENTION

The present invention relates to novel nucleic acids encoding RNA polymerase alpha subunit from M. tuberculosis and methods for use thereof.

BACKGROUND OF THE INVENTION

The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis in humans and is responsible for millions of deaths worldwide each year (Bloom et al. Science 257:1055, 1992). The emergence of multidrug resistant forms of tuberculosis has mandated the development of new antibiotics effective against refractory M. tuberculosis strains. Novel drugs which, like rifampin (Vall-Spinosa et al., N. Eng. J. Med. 283: 616, 1970), may be capable of inhibiting the prokaryotic transciptional machinery, could contribute significantly to the development of therapies to combat M. tuberculosis.

The RNA polymerase of eubacteria consists of four subunits (α, β, β′, and σ) and exists in two major forms: core enzyme (α₂, β, β′) and holoenzyme (α₂, β, β′ plus one of several σ subunits) (Chamberlin, in RNA Polymerase, Losick et al., eds., Cold Spring Harbor, 1976, pp. 17-67). The α subunit contains determinants for protein-protein interactions with transcription activators and protein-DNA interaction with upstream elements (Ishihama, Mol.Microbiol. 6:3283, 1992; Russo et al., J. Biol.Chem. 267:14515, 1992; Ebright et al., Curr.Opin.Genet.Dev. 5:197, 1995). The amino terminal domain of α is also required for assembly of the multisubunit core RNA polymerase (Ishihama, Adv. Biophys. 14:1, 1981). The β subunit is essential for transcript initiation and elongation, which β′ apparently functions in binding of the core enzyme to template DNA (Yura et al., Ann.Rev.Genet. 13:59, 1979). Core RNA polymerase is capable of RNA synthesis; however, σ is required for specific initiation of transcripts at promoter sequences (Gross et al., in Transcriptional Regulation, Cold Spring Harbor, 1992, pp. 129-176).

Discovery of inhibitors of M. tuberculosis RNA polymerase is hampered by a lack of information concerning components of the M. tuberculosis transcriptional apparatus, difficulties in obtaining sufficient yields of active enzymes for biochemical studies, and biosafety concerns. Establishment of an in vitro transcription system employing purified and reconstituted RNA polymerase would greatly advance efforts to identify new therapeutic agents active against tuberculosis.

Accordingly, there is a need in the art for compositions and methods utilizing cloned genes and purified proteins derived from M. tuberculosis RNA polymerase.

SUMMARY OF THE INVENTION

The present invention is based on the isolation and characterization of DNA encoding the a subunit of RNA polymerase derived from M. tuberculosis. In one aspect, the invention provides a purified, isolated nucleic acid having SEQ. I.D. No. 4 the sequence shown in FIG. 3. The invention also encompasses sequence-conservative and function-conservative variants of this sequence. The invention also provides vectors comprising these sequences, and cells comprising the vectors.

In another aspect, the present invention provides a purified, isolated polypeptide encoded by the nucleic acid SEQ. I.D. No. 5 sequence shown in FIG. 3, as well as function-conservative variants thereof. In one embodiment, the invention provides a purified α subunit further comprising a hexahistidine tag. The invention also provides purified, reconstituted core- and holoenzyme comprising the α subunit.

In yet another aspect, the invention provides in vitro methods for high-throughput screening to detect inhibitors of M. tuberculosis RNA polymerase. In one embodiment, the methods comprise:

a) providing a mixture comprising

(i) purified M. tuberculosis RNA polymerase and

(ii) a DNA template encoding a promoter sequence that is recognized by M. tuberculosis RNA polymerase;

b) incubating the mixture in the presence of test compounds to form test samples, and in the absence of test compounds to form control samples, under conditions that result in RNA synthesis in the control samples;

c) measuring RNA synthesis directed by said M. tuberculosis-recognized promoter in the test and control samples; and

d) comparing the RNA synthesis detected in step (c) between the test and control samples. According to the invention, an RNA polymerase inhibitor is a test compound that causes a reduction in RNA synthesis directed by the M. tuberculosis-recognized promoter measured in the test sample relative to RNA synthesis measured in the control sample. In a preferred embodiment, the purified RNA polymerase used in practicing this method comprises recombinant subunits which are reconstituted to form an enzymatically active holoenzyme. In another embodiment, the ability of test compounds to bind to purified α subunit is monitored.

In yet another aspect, the invention provides in vivo methods for high-throughput screening to detect inhibitors of M. tuberculosis RNA polymerase. The methods are carried out by the steps of:

a) providing a non-mycobacterial bacterial strain, preferably E. coli, that

(i) has been transformed with a DNA template encoding a promoter sequence that is recognized by M. tuberculosis RNA polymerase, and

(ii) expresses enzymatically active M. tuberculosis RNA polymerase (e.g., α₂, β, β′ plus one of several σ subunits);

b) incubating the bacterial strain of (a) in the presence of test compounds to form test samples, and in the absence of test compounds to form control samples;

c) measuring RNA synthesis directed by said M. tuberculosis-recognized promoter in the test and control samples; and

d) comparing the RNA synthesis directed by said M. tuberculosis-recognized promoter detected in step (c) between the test and control samples. According to the invention, an RNA polymerase inhibitor is a test compound that causes a reduction in RNA synthesis measured in the test sample relative to RNA synthesis measured in the control sample.

These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present specification and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic illustration comparing the deduced amino acid sequence of the M. tuberculosis rpoA gene fragment SEQ. I.D. No. 1 and corresponding regions of α subunits of B. subtilis and E. coli. Mt,SEQ. I.D. No. 3 M. tuberculosis; Bs, B. subtilis; Ec, E. coli. Dashed lines denote conserved segments of B. subtilis and E. coli proteins for which oligonucleotides were designed.

FIG. 2A is a photographic illustration of a Southern blot analysis of rpoA hybridizing cosmid clones. Four different positive clones (designated IIA5, IIA3, ID9, and IC5) isolated from an M. tuberculosis cosmid library were digested with NotI and hybridized with an rpoA gene probe. Sizes of DNA markers (λ EcoT141, Amersham) are indicated in kb.

FIG. 2B is a photographic illustration of a Southern blot analysis of rpoA hybridizing cosmid clones, showing structural conservation of genomic and cosmid-borne rpoA sequences. Purified cosmid IC5 DNA (lanes 1-5) or M. tuberculosis H37Rv genomic DNA (lanes 7-11) were digested with the indicated restriction enzymes and hybridized with an rpoA probe. Sizes of DNA markers (λ EcoT141, Amersham) are indicated in kb.

FIG. 3A and FIG. 3B shows the nucleotide SEQ. I.D. No.4 and deduced amino acid SEQ. I.D. No. 5 sequences of the M. tuberculosis H37Rv rpoA gene. RBS denotes a potential ribosome binding site. The putative start and stop codons of the rpoA gene product are at 85-87 and 1126-1128, respectively.

FIG. 4A and FIG. 4B is a graphic illustration comparing the inferred amino acid sequence of the M. tuberculosis H37Rv α subunit with SEQ. I.D. No. 4 sequences of α subunits from other bacteria. B. subtilis (Bs), SEQ. I.D. No. 6 M. genitalium (Mg), SEQ. I.D. No. _b 7 E. coli (Ec), SEQ. I.D. No. 8 H. influenza (Hi), SEQ. I.D. No. 9 B. pertusis (Bp), SEQ. I.D. No. 10 S. typhimurium (St), SEQ. I.D. No. 11 and C. trachomitis (Ct) SEQ. I.D. No. 12. Shading indicates identical amino acid residues. Amino acid sequence alignments were performed using MegAlign (DNAStar).

FIG. 5 is a photographic illustration of an SDS-polyacrylamide gel electropherogram depicting the expression and purification of M. tuberculosis a subunit in E. coli. Lane 1 shows molecular mass markers (kDa); lanes 2 and 3 show total proteins from uninduced and IPTG-induced cultures, respectively, of E. coli harboring pJH37; and lane 4 shows purified M. tuberculosis α subunit (approximately 38 kDa), which is indicated by an arrow.

FIG. 6 is a photographic illustration of an SDS-polyacrylamide gel electropherogram showing the reconstitution and purification of core- and holo-RNA polymerase from M. tuberculosis. Lanes 1 and 6 show molecular mass markers (kDa); lane 2 shows a-histidine-containing core enzyme; lane 3 shows a histidine-containing holoenzyme; lane 4 shows N-terminal histidine-tagged primary sigma; and lane 5 shows purified E. coli holoenzyme.

FIG. 7 is a graphic illustration of in vitro transcription reactions employing reconstituted purified M. tuberculosis RNA polymerase holoenzyme and T101 and T125 promoter-containing DNA templates.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited herein are hereby incorporated by reference in their entirety. In the case of inconsistencies the present disclosure will prevail.

The present invention is based on the isolation of a fragment of the M. tuberculosis rpoA gene, encoding the α subunit of RNA polymerase. As described in Example 1 below, the fragment was isolated by the polymerase chain reaction (PCR) using primers directed against regions of a that are highly conserved among Gram-positive and Gram-negative bacteria. The amplified DNA was utilized as a hybridization probe to recover the entire rpoA gene from a cosmid library of genomic DNA from virulent M. tuberculosis strain H37RV. Nucleotide sequencing indicated that the 1044 bp M. tuberculosis rpoA open reading frame (ORF) encodes a protein of 347 amino acids which shows significant structural similarity to the α subunits of diverse bacterial species with greatest identity to the B. subtilis α protein (FIG. 1).

Though structurally conserved throughout most of its length, the inferred amino acid sequence of the M. tuberculosis α subunit diverged from that of B. subtilis and other bacteria at its extreme C-terminus. Interestingly, studies on E. coli RNA polymerase subunits have indicated that the C-terminal 94 residues of α function in interactions with certain transcriptional regulatory proteins, while the N-terminal domain may play a more highly conserved role in facilitating the core assembly (Ishihama, Mol.Microbiol. 6:3283, 1992; Russo et al., J.Biol.Chem. 267:14515, 1992).

The B. subtilis rpoA gene is part of a large operon that is cotranscribed with genes for initiation factor 1 and ribosomal proteins B, S13, S11 , and L17; and, unlike the pattern of gene organization seen in E. coli the B. subtilis rpoA gene cluster is genetically linked to the rpoB-C operon encoding RNA polymerase components β and β′ (Boylan et al., J.Bacteriol. 171:2553, 1992).

In practicing the present invention, many techniques in molecular biology, microbiology, recombinant DNA, and protein biochemistry such as these explained fully in, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DNA Cloning: A Practical Approach, Volumes I and II, 1985 (D. N. Glover ed.); Oligonucleotide Synthesis, 1984, (M. L. Gait ed.); Transcription and Translation, 1984 (Hames and Higgins eds.); A Practical Guide to Molecular Cloning; the series, Methods in Enzymology (Academic Press, Inc.); and Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), may be used.

The present invention encompasses nucleic acid sequences encoding the α subunit of M. tuberculosis RNA polymerase, enzymatically active fragments derived therefrom, and related sequences. As used herein, a nucleic acid that is “derived from” a sequence refers to a nucleic acid sequence that corresponds to a region of the sequence, sequences that are homologous or complementary to the sequence, and “sequence-conservative variants” and “function-conservative variants”. Sequence-conservative variants are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position. Function-conservative variants are those in which a given amino acid residue in the α subunit has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar physico-chemical properties (such as, for example, acidic, basic, hydrophobic, and the like). Fragments of the α subunit that retain enzymatic activity can be identified according to the methods described herein, such as e.g., expression in E. coli followed by enzymatic assay of the cell extract or by purification and reconstitution of the purified polypeptide into an enzymatically active holoenzyme.

The nucleic acids of the present invention include purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotides or mixed polyribo-polydeoxyribo nucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases. The nucleic acids may be isolated directly from cells. Alternatively, PCR can be used to produce the nucleic acids of the invention, using either chemically synthesized strands or genomic material as templates. Primers used for PCR can be synthesized using the sequence information provided herein and can further be designed to introduce appropriate new restriction sites, if desirable, to facilitate incorporation into a given vector for recombinant expression.

The nucleic acids of the present invention may be flanked by natural M. tuberculosis regulatory sequences, or may be associated with heterologous sequences, including promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5′- and 3′-noncoding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acids may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The nucleic acid may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the nucleic acid sequences of the present invention may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.

The invention also provides nucleic acid vectors comprising the disclosed α subunit sequences or derivatives or fragments thereof. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clontech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), or pRSET or pREP (Invitrogen, San Diego, Calif.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes. Suitable host cells may be transformed/transfected/infected as appropriate by any suitable method including electroporation, CaCl₂ mediated DNA uptake, fungal infection, microinjection, microprojectile, or other established methods.

Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are E. coli, B. subtilis, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Schizosaccharomyces pombi, SF9 cells, C129 cells, 293 cells, Neurospora. and CHO cells, COS cells, HeLa cells, and immortalized mammalian myeloid and lymphoid cell lines. Preferred replication systems include M13, ColE1, SV40, baculovirus, lambda, adenovirus, and the like. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner of manipulation, etc. are known in the art. Under appropriate expression conditions, host cells can be used as a source of recombinantly produced Mycobacterial-derived peptides and polypeptides.

Advantageously, vectors may also include a transcription regulatory element (i.e., a promoter) operably linked to the α subunit portion. The promoter may optionally contain operator portions and/or ribosome binding sites. Non-limiting examples of bacterial promoters compatible with E. coli include: trc promoter, β-lactamase (penicillinase) promoter; lactose promoter; tryptophan (trp) promoter; arabinose BAD operon promoter; lambda-derived P1 promoter and N gene ribosome binding site; and the hybrid tac promoter derived from sequences of the trp and lac Uv5 promoters. Non-limiting examples of yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase (GALI) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter. Suitable promoters for mammalian cells include without limitation viral promoters such as that from Simian Virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus (ADV), and bovine papilloma virus (BPV). Mammalian cells may also require terminator sequences and poly A addition sequences, and enhancer sequences which increase expression may also be included. Sequences which cause amplification of the gene may also be desirable. Furthermore, sequences that facilitate secretion of the recombinant product from cells, including, but not limited to, bacteria, yeast, and animal cells, such as secretory signal sequences and/or prohormone pro region sequences, may also be included.

Nucleic acids encoding wild-type or variant α subunit polypeptides may also be introduced into cells by recombination events. For example, such a sequence can be introduced into a cell, and thereby effect homologous recombination at the site of an endogenous gene or a sequence with substantial identity to the gene. Other recombination-based methods, such as non-homologous recombinations or deletion of endogenous genes by homologous recombination, may also be used.

α subunit-derived polypeptides according to the present invention, including function-conservative variants, may be isolated from wild-type or mutant M. tuberculosis cells, or from heterologous organisms or cells (including, but not limited to, bacteria, fungi, insect, plant, and mammalian cells) into which an α subunit-derived protein-coding sequence has been introduced and expressed. Furthermore, the polypeptides may be part of recombinant fusion proteins (see, e.g., Example 2 below). Alternatively, polypeptides may be chemically synthesized by commercially available automated procedures, including, without limitation, exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis.

“Purification” of an α subunit polypeptide refers to the isolation of the polypeptide in a form that allows its enzymatic activity to be measured without interference by other components of the cell in which the polypeptide is expressed. Methods for polypeptide purification are well-known in the art, including, without limitation, preparative disc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phase HPLC, gel filtration, ion exchange and partition chromatography, and countercurrent distribution. For some purposes, it is preferable to produce the polypeptide in a recombinant system in which the protein contains an additional sequence tag that facilitates purification, such as, but not limited to, a polyhistidine sequence. The polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix. Alternatively, antibodies produced against the α subunit or against peptides derived therefrom can be used as purification reagents. Other purification methods are possible.

The isolated polypeptides may be modified by, for example, phosphorylation, sulfation, acylation, or other protein modifications. They may also be modified with a label capable of providing a detectable signal, either directly or indirectly, including, but not limited to, radioisotopes and fluorescent compounds.

Screening Methods to Identify Anti-tuberculosis Agents

The methods and compositions of the present invention can be used to identify compounds that inhibit the function of M. tuberculosis RNA polymerase and thus are useful as anti-tuberculosis agents. This is achieved by providing enzymatically active recombinant α subunit according to the present invention, in combination with other components of RNA polymerase, in a context in which the inhibitory effects of test compounds can be measured. Alternatively, the ability of test compounds to bind to purified α subunit is monitored, using methods well-known in the art. Test compounds identified using the methods of the invention encompass those that interfere with the enzymatic activity of RNA polymerase and/or disrupt the interaction of the α subunit with transcriptional activators. Thus, the compounds may function as general or gene-specific transcriptional inhibitors.

In a preferred embodiment, recombinant M. tuberculosis RNA polymerase subunits (α, β, β′ plus one of several α subunits) are purified in milligram quantities from E. coli cultures by affinity methods utilizing a hexahistidine tagged α subunit. Enzymatically active holoenzyme is reconstituted using these components (see, e.g., Example 2 below). The active polymerase is then incubated in the presence of test compounds to form test mixtures, and in the absence of test compounds to form control mixtures. ln vitro transcription is then carried out using a DNA template containing appropriate promoter sequences (see, e.g., Example 2 below).

In another embodiment, M. tuberculosis RNA polymerase subunits (α₂, β, β′ plus one of several σ subunits) are co-expressed in E. coli or another surrogate bacterial cell, in conjunction with an appropriate promoter functionally linked to a reporter gene. The ability of test compounds to differentially inhibit M. tuberculosis RNA polymerase is then assessed by measuring the amount and/or activity of the reporter gene product.

M. tuberculosis promoters useful in practicing the invention include without limitation: P1 or P2 hsp 60 promoters (Stover et al., Nature 351:456, 1991); cpn-60 promoter (Kong et al., Proc.Natl.Acad.Sci. USA 90:2608, 1993); 85A antigen promoter (Kremer, J. Bacteriol. 177:642, 1995); PAN promoter (Murray et al., Mol. Microbiol. 6:3331, 1992); 16S RNA promoter (Ji et al., Microbiol. 140: 2829, 1994); and askβ promoter (Cirillo et al., Mol. Microbiol. 11:629, 1994). Useful reporter genes include without limitation xylE (Curcic et al., Mol. Microbiol 13:1057, 1994); CAT (Das Gupta et al., J. Bacteriol. 175:5186, 1993); luciferase (Cooksey et al., Antimicrob.Agts.Chemother. 37:1348, 1993); green fluorescent protein (Dhadayuthap et al., Mol. Microbiol. 17:901, 1995); and lacZ (Silhavy et al., Microbiol.Rev. 49:398, 1985).

It will be understood that the present invention encompasses M. tuberculosis RNA polymerases containing any appropriate a factor, which is used in conjunction with a particular promoter that is recognized by RNA polymerase containing that σ factor. Non-limiting examples of useful σ factors include σ^(A), σ^(B), and σ^(F) (Doushkan et al., 1995; DeMaio et al., Gene 165:67, 1995). The invention also encompasses the identification of additional promoters that are recognized by a particular σ subunit. This is achieved by providing a library of random M. tuberculosis gene fragments cloned upstream of an appropriate reporter gene (see above). The library is transformed into M. tuberculosis or M. smegmatis and reporter gene expression is measured. Alternatively, the library is transformed into another bacterial cell, such as, e.g., E. coli, which expresses M. tuberculosis RNA polymerase core subunits as well as a σ subunit of interest and cognate promoters that drive reporter gene expression. In yet another embodiment, expression of an M. tuberculosis σ factor confers new recognition properties on E. coli RNA polymerase and permits isolation of promoters utilized specifically by a particular M. tuberculosis σ subunit.

Preferably, both in vitro and in vivo screening methods to detect M. tuberculosis-specific RNA polymerase inhibitors are adapted to a high-throughput format, allowing a multiplicity of compounds to be tested in a single assay. Such inhibitory compounds may be found in, for example, natural product libraries, fermentation libraries (encompassing plants and microorganisms), combinatorial libraries, compound files, and synthetic compound libraries. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., TibTech 14:60, 1996). preferably using automated equipment, to allow for the simultaneous screening of a multiplicity of test compounds.

Useful anti-tuberculosis compounds are identified as those test compounds that decrease tuberculosis-specific transcription. Once a compound has been identified by the methods of the present invention as an RNA polymerase inhibitor, in vivo and in vitro tests may be performed to further characterize the nature and mechanism of the inhibitory activity. For example, classical enzyme kinetic plots can be used to distinguish, e.g., competitive and non-competitive inhibitors.

Compounds identified as RNA polymerase inhibitors using the methods of the present invention may be modified to enhance potency, efficacy, uptake, stability, and suitability for use in pharmaceutical formulations, etc. These modifications are achieved and tested using methods well-known in the art.

The present invention is further described in the following examples which are intended to further describe the invention without limiting the scope thereof.

EXAMPLE 1

Isolation and Analysis of the M. tuberculosis rpoA Gene

The following experiments were performed in order to clone the gene encoding M. tuberculosis RNA polymerase α subunit. In these experiments, the following materials and methods were used.

A. Materials and Methods

PCR amplification: M. tuberculosis H37Rv genomic DNA (1 ng) was amplified in a 100 μl reaction containing: 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂, 500 mM KCl, 2.5 U Taq polymerase (Beohringer-Mannheim), 0.2 mM dNTPs and oligonucleotide primers (1 μM each): 5′-CTACGCAAGCAGGGTCCGGGTGAG-3′ and 5′-CTCACCCGGACCCTGCTTGCGTAG-3′. PCR was performed in an Omnigene thermocycler with 35 cycles of 94° C. for 1 min, 55° C. for 2 min, 72° C. for 1 min. The amplification product was gel-purified and ligated into vector pCR2.1 (Invitrogen).

Cosmid hybridizations: A transducing lysate of a cosmid library of M tuberculosis H37Rv genomic DNA in vector pYA3060 was used. Cosmid-bearing E. coil Φ2819T (Jacobs et al., Infec.Immun. 52:101, 1986) colonies representing roughly five genomic equivalents were individually picked to wells of sterile 96-well microliter dishes and propagated at 30° C. in Luria broth containing ampicillin at 30 μg/ml and thymidine at 50 μg/ml. Colonies were grown overnight at room temperature on the above media as nylon filter replicas of the library. Filters were processed for colony hybridization by standard methods and probe hybridizations performed as described below. Cosmid DNAs were purified using maxiprep columns (Qiagen).

Southern blot analysis: Restriction enzyme digests of M. tuberculosis H37Rv chromosomal or cosmid DNAs were resolved by electrophoresis on 1% TAE-agarose gels and blotted to nylon filters using standard methods. Probe labeling and hybridizations were performed using the Gene Images hybridization system (Amersham) essentially as described by the supplier except that hybridizations and washes were carried out at 70° C.

DNA sequencing and analysis: Plasmid templates for nucleotide sequencing were purified using RPM miniprep kits (Bio101). PCR cycle sequencing (ABI Prizm) was carried out with an Applied Biosystems automated sequencer.

B. Strategy for M. tuberculosis rpoA Gene Isolation

A fragment of the M. tuberculosis rpoA gene was isolated by PCR, using oligonucleotide primers SEQ. I.D. Nos. 13 and 14 homologous to defined regions of α that are conserved among Gram-positive and Gram-negative bacterial species. Amino acid sequence alignments of the α subunits of B. subtilis and E. coli SEQ. I.D. Nos. 2 and 3, revealed several short regions of identity clustered primarily within the N-terminal half of α implicated in association with RNA polymerase core subunits. Primers corresponding to conserved a segments LRRILLSS and VTAADITHD (amino acids 39-46 and 106-114, respectively of the B. subtilis subunit) specifically amplified a DNA fragment of anticipated size (228 bp) using M. tuberculosis H37Rv genomic DNA as a template. To confirm that the amplified DNA contained rpoA sequences, the 228 bp fragment was subcloned and a partial nucleotide sequence determined. The deduced amino acid sequence of the PCR product showed SEQ. I.D. No. 1 significant homology to the α subunits of other bacteria SEQ. I.D. Nos. 2 and 3 (FIG. 1), confirming its identity as an rpoA gene fragment. One ORF of the 228 bp fragment displayed high degree of identity to corresponding intervals of the B. subtilis (61%) and E. coli (55%) α subunits. The M. tuberculosis rpoA-derived sequence had an overall G+C content of 60% while the G+C content for bases occupying the third position of each codon increased to 76%, values in good agreement with compositions of other M. tuberculosis genes (Weiden et al., in Tuberculosis, Little, Brown, 1996, pp. 211-222).

C. Southern Blot Analvsis and Isolation of the Entire M. tuberculosis rpoA Gene

To establish that the cloned 228 bp fragment was mycobacterial in origin, Southern blot analysis of restriction enzyme digests of M. tuberculosis H37Rv genomic DNA was performed. The PCR probe detected unique restriction fragments in chromosomal digests (FIG. 2B). These data are consistent with a single copy of the rpoA gene in the M. tuberculosis chromosome (two bands of hybridization observed in the EcoRI digest result from the presence of an EcoRI site within the 228 bp probe sequence). The full-length rpoA gene was obtained from a cosmid library of M. tuberculosis H37Rv genomic fragments using the 228 bp PCR probe. Screening of approximately 600 cosmid-bearing E. coli colonies (representing roughly 5 genome equivalents) with the rpoA gene fragment yielded 4 positive clones. Restriction analysis indicated that the 4 isolates differed from one another with respect to total insert size, though all shared a roughly 10 kb NotI fragment which hybridized to the rpoA gene probe (FIG. 2A). One rpoa-hybridizing cosmid clone, designated IC5, was chosen for further analysis. Southern blotting of IC5 DNA digested with a panel of restriction enzymes confirmed that the no gross structural rearrangements of the rpoA gene had occurred during cloning (FIG. 2B). The 1.4 kb SacII and 1.3 kb SaII rpoA-hybridizing fragments of cosmid IC5 were subcloned into vector pSKII+ prior to nucleotide sequencing.

D. Sequence Analysis of the M. tuberculosis rpoA Gene

Nucleotide sequencing was performed on plasmid subclones and on cosmid IC5 DNA. Nucleotide and deduced amino acid SEQ. I.D. Nos. 4 and 5 respectively sequences of the rpoA gene of M. tuberculosis are shown in FIGS. 3A and 3B. The sequence encodes a 1044 bp ORF which has an overall G+C composition of 63% (85% for bases occupying the codon third position). Assuming that the ATG at position 85-87 serves as the initiator codon, the ORF is expected to encode a protein of 347 amino acids. A strong match with the consensus sequence for an M. tuberculosis ribosome binding site (GAAAGGA), (Novick, in Tuberculosis, Little, Brown, 1996, pp. 187-198) is positioned just upstream of the putative ATG codon. Examination of more than 80 bp of nucleotide sequence upstream of the translation start site did not reveal regions of exact identity with prokaryotic promoter sites. Among α subunits studied in other bacterial species, the deduced amino acid sequence of the 347 residue M. tuberculosis protein SEQ. I.D. No. 5 displayed greatest similarity (48% identity) to the α subunit of the Gram-positive sporulating bacterium B. subtilis SEQ. I.D. No. 6 (FIGS. 4A and 4B).

EXAMPLE 2

Expression of M. tuberculosis RNA Polymerase α Subunit and Reconstitution of Active M. tuberculosis RNA Polymerase

The following experiments were performed to purify M. tuberculosis RNA polymerase α subunit that had been expressed in E. coli and to reconstitute enzymatically active RNA polymerase holoenzyme.

A. Methods

Purification of M. tuberculosis α subunit: Recombinant α was produced in E. coil BL21(DE3) cells (Novagen) and grown at 37° C. in LB medium containing 25 μg/ml/kanamycin. Overnight cultures (100 ml) were diluted 10-fold and grown in LB medium containing 25 μg/ml kanamycin until A₆₀₀=0.6 (approximately 75 min). Expression of α was induced for 3 hr with 4 mM IPTG. Purification of the α subunit was performd essentially as described by Tang, et. al, Meth.Enzymol. 273:130, 1996. Briefly, cell pellets were harvested by centrifugation for 10 min At 4° C. (3000×g). The pellet was resuspended in 20 ml buffer B (6 M guanidine HCl, 10 mM Tris (pH 7.9), 500 mM NaCl, 5 mM imidazole) and lysed by sonication. Inclusion bodies were removed by centrifugation at 42,000×g for 30 min at 4° C. The sample was then adsorbed onto Ni²⁺-NTA (Qiagen) in buffer B. The sample was washed twice with 20 ml buffer B; washed twice with buffer B containing 30 mM imidazole; and eluted with 10 ml buffer B containing 500 mM imidazole. Adsorption, washes, and elution were performed with 1 min incubations at 4° C. with gentle mixing.

Reconstitution of α with other M. tuberculosis subunits to form core/holo-RNA polymerase: Reconstitution of core/holo-enzyme was performed by combining 60, 300, and 600 μg of α, β, and β′, respectively, followed slow removal of the denaturant by dialysis. The combined protein concentration was adjusted to 0.5 mg/ml with buffer B to prevent aggregation (Nobuyuki et al., Meth.Enzymol 273:121, 1996) and dialysed against buffer E (50 mM Tris (pH 7.9), 200 mM KCl, 10 mM MgCl₂, 10 μM ZnCl₂, 1 mM EDTA, 5 mM β-mercaptoethanol, 20% (v/v) glycerol) overnight at 4° C. Following dialysis, the sample was activated by incubation for 45 min at 30° C. and aggregates cleared by centrifugation at 10,000×g for 10 min at 4° C. The sample was adsorbed onto Ni²⁺-NTA in buffer F (50 mM Tris (pH 7.9), 0.5 mM EDTA, 5% (v/v) glycerol), and washed three times in buffer F containing 5 mM imidazole, and eluted in buffer F containing 150 mM imidazole. Adsorption and elution were performed by incubating for 45 min at 4° C. and washes for 1 min. Each step was followed by centrifugation at 16,000×g for 2 min at 4° C. Core/holo-enzyme was dialysed in buffer F (50 mM Tris (pH 7.9), 50 mM KCl, 0.5 mM EDTA, 5% (v/v) glycerol) overnight at 4° C.

Purification of M. tuberculosis holoenzyme: Core polymerase and subassemblies were separated from holoenzyme by elution from a MonoQ column (Pharmacia) using a 0.2-0.5 M KCl gradient. Fractions containing holoenzyme were pooled and used the in vitro transcription assay.

Promoter construction: Recently, mycobacterium promoters have been cloned using a plasmid shuttle vector pSD7 (Das Gupta et al., J. Bacteriol. 175:5186, 1993) and in a later study, the strength of these promoters was analyzed (Bashyam et al., J. Bacteriol. 178:4847, 1996). Based on these studies two promoters, T125 and T101, identified as a weak and a strong promoter, respectively, were cloned into the pUC19 vector and used as templates for M. tuberculosis transcription asays.

Transcriptional activity: Holoenzyme was reconstituted by the addition of primary sigma factor MysA to the core polymerase and incubation at 30° C. for 20 minutes. The transcription reaction was performed as described (Shorenstein et al., J. Biol.Chem. 248:6170, 1973) except that 3 μg of template (pUC19, pMC116, or pMC117) was used per reaction containing a high salt transcription buffer (50 mM Tris (pH7.9), 10 MgCl₂, 200 mM KCl, 10 mM DTT, 0.1 mM EDTA, 1 mM K₂HPO₄ (pH7.5), 100 μg/ml BSA). 50 μl reactions were incubated for 30 minutes at 37° C. To precipitate the RNA transcripts and to stop the reaction, 100 μl of 10% TCA were added to the reaction. The TCA-precipitated RNA was adsorbed onto UniFilter GF/C (Packard, Meriden, Conn.) double-thick glass fiber filtermats using a cell harvester (Packard, Meriden, Conn.). The wells of the microtiter plate and the filter were washed two times with 5% TCA and bound radioactivity was determined using a TopCount-HTS (Packard, Meriden, Conn.) scintillation counter.

B. Results

Recombinant M. tuberculosis α subunit was overproduced in soluble form in E. coli cells transformed with plasmid pJH37, a derivative of pET26b, containing the entire rpoA coding region. M. tuberculosis α was expressed at high levels as a C-terminal hexahistidine-tagged fusion protein and purified to homogeneity by affinity chromatography. The majority of the 6xHis-tagged α subunit was located in the soluble fraction and was further purified using a Ni²⁺-NTA column. Elution of the α subunit polypeptide was performed using 0.5 M imidazole, resulting in >90% recovery of the subunits with >95% purity as estimated by SDS-PAGE (FIG. 5).

Core enzyme was reconstituted by combining crude preparations of the β and β′ subunits with α polypeptide that contained a histidine tag, followed by removal of denaturant by dialysis to facilitate refolding. Holoenzyme was reconstituted by combining the core polymerase with purified primary σ (MysA) from M. tuberculosis. The core/holoenzyme, containing a 6xHis tag on the α subunit, was purified from free subunits and subassemblies by batch elution from Ni²⁺-NTA followed by elution from ion exchange chromatography (FIG. 6).

To demonstrate that reconstituted M. tuberculosis RNA polymerase possesses transcriptional activity, in vitro transcription reactions were performed using MysA-containing recombinant holoenzyme and templates containing two known MysA-dependent promoters (T101 and T125, which correspond to strong and weak mycobacterial promoters, respectively). The transcriptional activity of the reconstituted holoenzyme was more than two-fold higher when T101 was used as template as compared with T125 (FIG. 7), suggesting that the rate of transcription was related to both the strength of the promoter and to the particular σ factor used.

These experiments demonstrated that M. tuberculosis α subunit produced according to the invention can be purified and reconstituted into enzymatically active RNA polymerase holoenzyme that exhibits specificity for M. tuberculosis promoters.

EXAMPLE 3

High Throughput Screens for Inhibitors of M. Tuberculosis RNA Polymerase and α Subunit

High-throughput screens for anti-tuberculosis agents may be performed using either an in vitro or in vivo format. In either case, the ability of test compounds to inhibit M. tuberculosis RNA polymerase-driven transcription of M. tuberculosis promoters is tested. Typically, a strong promoter such as T101 is used for the test reactions. The sequence of the T101 promoter is: identified by SEQ. ID. NO. 15 and read as follows.

T101:5′

AGCTTGCAGATCTAGCGATCGCAGCCGACGTGATACCTGACCGTTGTTGATA GTGTCGGCGGCT-3′

a) In vitro screens:

The following procedure is used for cell-free high-throughput screening. A Tomtec Quadra 96-well pipetting station is used to add the reaction components to polypropylene 96-well dishes. 5 μl aliquots of test compounds dissolved in DMSO (or DMSO alone as a control) are added to wells. This is followed by 20 μl of the RNA polymerase mixture, which consists of: 10 mM DTT, 200 mM KCl, 10 mM Mg⁺², 1.5 μM bovine serum albumin, and 0.25 μg reconstituted RNA polymerase. After allowing the test compound to interact with the RNA polymerase, 25 μl of the DNA/NTP mixture is added, containing: 1 μg template DNA (see above), 4 μM [α-³²P]-UTP, and 400 μM each CTP, ATP, and GTP.

After incubation for 30 min at 25° C., the reaction is stopped by addition of 150 μl 10% trichloroacetic acid (TCA). After incubation at room temperature for 60 min, the TCA-precipitated RNA is adsorbed onto double-thick glass fiber filtermats using a Tomtec cell harvester. The wells of the microtiter plate and the filter are washed twice with 5% TCA and bound radioactivity is determined usinjg a Wallac microbeta 1450 scintillation counter. Inhibitory activity due to the test compound is calculated according to the formula: ${\% \quad {inhibition}} = {\frac{\left( {{cpm}_{{positive}\quad {control}} - {cpm}_{sample}} \right)}{{cpm}_{{positive}\quad {control}}} \times 100}$

where cpm_(positive control) represents the average of the cpm in wells that received DMSO alone, and cpm_(sample) represents the cpm in the well that received test compound. Compounds that cause at least 50% inhibition are scored as positive “hits” in this assay.

As an additional control, rifampicin is used at a concentration of 30 nM, which results in a 50-75% inhibition of transcription in this assay.

b) In vivo screen:

M. tuberculosis RNA polymerase subunits (α, β, β′, and a particular σ subunit) are expressed in E. coli under the control of regulatable promoters by transforming E. coli with appropriate plasmids. If the σ^(A) subunit is expressed, a DNA sequence comprising the T101 promoter is also introduced into the cells to serve as a template for M. tuberculosis-specific transcription.

In one embodiment, the T101 promoter sequence is linked to a DNA sequence encoding the xylE gene product, catechol 2, 3-dioxygenase (CDO). When expressed in the E. coli cell, CDO converts catechol to 2-hydroxymuconic semialdehyde, which has a bright yellow color (having an absorbance maximum at 375 nm) that is easily detected in whole cells or in crude extracts. The substrate for this enzyme is a small aromatic molecule that easily enters the bacterial cytoplasm and does not adversely affect cell viability.

In a high-throughput format, aliquots of bacterial cultures are incubated in the absence or presence of test compounds, and CDO activity is monitored by measuring absorbance at 375 nm following addition of catechol.

c) Specificity:

Compounds that score as positive in either the in vitro or in vivo assays described above are then tested for their effect on human RNA polyrnerase II. Those compounds which do not significantly inhibit human RNA polymerase II will be further developed as potential anti-tuberculosis agents.

15 1 59 PRT M. tuberculosis 1 Ile Pro Gly Ala Ala Val Thr Ser Ile Arg Ile Asp Gly Val Leu His 1 5 10 15 Glu Phe Thr Thr Val Pro Gly Val Lys Glu Asp Val Thr Glu Ile Ile 20 25 30 Leu Asn Leu Lys Ser Leu Val Val Ser Ser Glu Glu Asp Glu Pro Val 35 40 45 Thr Met Tyr Leu Arg Lys Gln Gly Pro Gly Glu 50 55 2 76 PRT B. subtilis 2 Leu Arg Arg Ile Leu Leu Ser Ser Leu Pro Gly Ala Ala Val Thr Ser 1 5 10 15 Ile Gln Ile Asp Gly Val Leu His Glu Phe Ser Thr Ile Glu Gly Val 20 25 30 Val Glu Asp Val Thr Thr Ile Ile Leu His Ile Lys Lys Leu Ala Leu 35 40 45 Lys Ile Tyr Ser Asp Glu Glu Lys Thr Leu Glu Ile Asp Val Gln Gly 50 55 60 Glu Gly Thr Val Thr Ala Ala Asp Ile Thr His Asp 65 70 75 3 76 PRT E. coli 3 Leu Arg Arg Ile Leu Leu Ser Ser Met Pro Gly Cys Ala Val Thr Glu 1 5 10 15 Val Glu Ile Asp Gly Val Leu His Glu Tyr Ser Thr Lys Glu Gly Val 20 25 30 Gln Glu Asp Ile Leu Glu Ile Leu Leu Asn Leu Lys Gly Leu Ala Val 35 40 45 Arg Val Gln Gly Lys Asp Glu Val Ile Leu Thr Leu Asn Lys Ser Gly 50 55 60 Ile Gly Pro Val Thr Ala Ala Asp Ile Thr His Asp 65 70 75 4 1230 DNA M. tuberculosis 4 cgagtacccc caccttcggg ggcgccgccc ccgagtgccc ccacagacgt catatggcgg 60 acgtcgaaag gaagaagaaa caccatgctg atctcacagc gccccaccct gtccgaggac 120 gtcctcaccg acaaccgatc ccagttcgtg atcgaaccgc tggagccggg attcggctac 180 accctgggca attcgctgcg tcgcaccctg ctgtcgtcga ttcccggagc ggccgtcacc 240 agcattcgca tcgatggtgt acgcaccctg ctgtcgtcga ttcccggagc ggccgtcacc 300 agcattcgca tcgatggtgt actgcacgaa ttcaccacgg tgcccggggt caaagaagat 360 gtcaccgaga tcatcctgaa tctcaagagc ctggtggtgt cctcggagga ggacgagccg 420 gtcaccatgt acctacgcaa gcagggtccg ggtgaggtta ccgccggcga catcgtgccg 480 ccggccggcg tcaccgtgca caaccccggc atgcacatcg ccacgctgaa cgataagggc 540 aagctggaag tcgagctcgt cgtcgagcgt ggccgcggct atgtcccggc ggtgcaaaac 600 cgggcttcgg gtgccgaaat tgggcgcatt ccagtcgatt ccatctactc accggtgctc 660 aaagtgacct acaaggtgga cgccacccgg gtcgagcagc gcaccgactt cgacaagctg 720 atcctggacg tggagaccaa gaattcaatc agcccgcgcg acgcgctggc gtcggctggc 780 aagacgctgg tcgagttgtt cggcctggca cgggaactca acgtcgaggc cgaaggcatc 840 gagatcgggc cgtcgccggc cgaggccgat cacattgcgt cattcgccct gccgatcgac 900 gacctggatc tgacggtgcg gtcctacaac tgcctcaagc gcgagggggt gcacaccgtg 960 ggcgaactgg tggcgcgcac cgaatccgac ctgcttgaca tccgcaactt cggtcagaag 1020 tccatcgacg aggtgaagat caagctgcac cagctgggcc tgtcactcaa ggacagcccg 1080 ccgagcttcg acccctcgga ggtcgcgggc tacgacgtcg ccaccggcac ctggtcgacc 1140 gagggcgcgt acgacgagca ggactacgcc gaaaccgaac agctttagac tgcctctaat 1200 ccagacagga gcgtcagcta tgcccaagcc 1230 5 347 PRT M. tuberculosis 5 Met Leu Ile Ser Gln Arg Pro Thr Leu Ser Glu Asp Val Leu Thr Asp 1 5 10 15 Asn Arg Ser Gln Phe Val Ile Glu Pro Leu Glu Pro Gly Phe Gly Tyr 20 25 30 Thr Leu Gly Asn Ser Leu Arg Arg Thr Leu Leu Ser Ser Ile Pro Gly 35 40 45 Ala Ala Val Thr Ser Ile Arg Ile Asp Gly Val Leu His Glu Phe Thr 50 55 60 Thr Val Pro Gly Val Lys Glu Asp Val Thr Glu Ile Ile Leu Asn Leu 65 70 75 80 Lys Ser Leu Val Val Ser Ser Glu Glu Asp Glu Pro Val Thr Met Tyr 85 90 95 Leu Arg Lys Gln Gly Pro Gly Glu Val Thr Ala Gly Asp Ile Val Pro 100 105 110 Pro Ala Gly Val Thr Val His Asn Pro Gly Met His Ile Ala Thr Leu 115 120 125 Asn Asp Lys Gly Lys Leu Glu Val Glu Leu Val Val Glu Arg Gly Arg 130 135 140 Gly Tyr Val Pro Ala Val Gln Asn Arg Ala Ser Gly Ala Glu Ile Gly 145 150 155 160 Arg Ile Pro Val Asp Ser Ile Tyr Ser Pro Val Leu Lys Val Thr Tyr 165 170 175 Lys Val Asp Ala Thr Arg Val Glu Gln Arg Thr Asp Phe Asp Lys Leu 180 185 190 Ile Leu Asp Val Glu Thr Lys Asn Ser Ile Ser Pro Arg Asp Ala Leu 195 200 205 Ala Ser Ala Gly Lys Thr Leu Val Glu Leu Phe Gly Leu Ala Arg Glu 210 215 220 Leu Asn Val Glu Ala Glu Gly Ile Glu Ile Gly Pro Ser Pro Ala Glu 225 230 235 240 Ala Asp His Ile Ala Ser Phe Ala Leu Pro Ile Asp Asp Leu Asp Leu 245 250 255 Thr Val Arg Ser Tyr Asn Cys Leu Lys Arg Glu Gly Val His Thr Val 260 265 270 Gly Glu Leu Val Ala Arg Thr Glu Ser Asp Leu Leu Asp Ile Arg Asn 275 280 285 Phe Gly Gln Lys Ser Ile Asp Glu Val Lys Ile Lys Leu His Gln Leu 290 295 300 Gly Leu Ser Leu Lys Asp Ser Pro Pro Ser Phe Asp Pro Ser Glu Val 305 310 315 320 Ala Gly Tyr Asp Val Ala Thr Gly Thr Trp Ser Thr Glu Gly Ala Tyr 325 330 335 Asp Glu Gln Asp Tyr Ala Glu Thr Glu Gln Leu 340 345 6 315 PRT B. subtilis 6 Met Ile Glu Ile Glu Lys Pro Lys Ile Glu Thr Val Glu Ile Ser Asp 1 5 10 15 Asp Ala Lys Phe Gly Lys Phe Val Val Glu Pro Leu Glu Arg Gly Tyr 20 25 30 Gly Thr Thr Leu Gly Asn Ser Leu Arg Arg Ile Leu Leu Ser Ser Leu 35 40 45 Pro Gly Ala Ala Val Thr Ser Ile Gln Ile Asp Gly Val Leu His Glu 50 55 60 Phe Ser Thr Ile Glu Gly Val Val Glu Asp Val Thr Thr Ile Ile Leu 65 70 75 80 His Ile Lys Lys Leu Ala Leu Lys Ile Tyr Ser Asp Glu Glu Lys Thr 85 90 95 Leu Glu Ile Asp Val Gln Gly Glu Gly Thr Val Thr Ala Ala Asp Ile 100 105 110 Thr His Asp Ser Asp Val Glu Ile Leu Asn Pro Asp Leu His Ile Ala 115 120 125 Thr Leu Gly Glu Asn Ala Ser Phe Arg Val Arg Leu Thr Ala Gln Arg 130 135 140 Gly Arg Gly Tyr Thr Pro Ala Asp Ala Asn Lys Arg Asp Asp Gln Pro 145 150 155 160 Ile Gly Val Ile Pro Ile Asp Ser Ile Tyr Thr Pro Val Ser Arg Val 165 170 175 Ser Tyr Gln Val Glu Asn Thr Arg Val Gly Gln Val Ala Asn Tyr Asp 180 185 190 Lys Leu Thr Leu Asp Val Trp Thr Asp Gly Ser Thr Gly Pro Lys Glu 195 200 205 Ala Ile Ala Leu Gly Ser Lys Ile Leu Thr Glu His Leu Asn Ile Phe 210 215 220 Val Gly Leu Thr Asp Glu Ala Gln His Ala Phe Ile Met Val Glu Lys 225 230 235 240 Glu Glu Asp Gln Lys Glu Lys Val Leu Glu Met Thr Ile Glu Glu Leu 245 250 255 Asp Leu Ser Val Pro Ser Tyr Asn Cys Leu Lys Arg Ala Gly Ile Asn 260 265 270 Thr Val Gln Glu Leu Ala Asn Lys Thr Phe Glu Asp Met Met Lys Val 275 280 285 Arg Asn Leu Gly Arg Lys Ser Leu Glu Glu Val Lys Ala Lys Leu Glu 290 295 300 Glu Leu Gly Leu Gly Ile Leu Arg Lys Asp Asp 305 310 315 7 328 PRT M. genitalium 7 Met Glu Lys Phe Leu Lys Tyr Glu Ile Lys Val Asn Asn Asn Gln Pro 1 5 10 15 Thr Asn Thr Asn Pro Asn Tyr Gly Ile Phe Glu Val Ala Pro Leu Glu 20 25 30 Ser Gly Phe Gly Ile Thr Ile Gly Asn Ala Met Arg Arg Val Leu Leu 35 40 45 Ser Cys Ile Pro Gly Ala Ser Val Phe Ala Ile Ala Ile Ser Gly Val 50 55 60 Lys Gln Glu Phe Ser Asn Val Glu Gly Val Leu Glu Asp Val Thr Glu 65 70 75 80 Met Val Leu Asn Phe Lys Gln Leu Val Val Arg Ile Ser Asp Leu Leu 85 90 95 Phe Glu Asp Gly Glu Met Ile Glu Pro Pro Leu Glu Arg Trp Pro Val 100 105 110 Leu Lys Val Thr Ala Glu Lys Lys Gly Ala Val Tyr Ala Lys Asp Leu 115 120 125 Glu Cys Pro Ala Gly Phe Glu Val Ile Asn Lys Asp Leu Tyr Leu Phe 130 135 140 Ser Leu Gln Lys Asp Met Lys Leu Thr Val Ser Val Tyr Val Lys Gln 145 150 155 160 Gly Arg Gly Phe Thr Ser Phe Leu Glu Asn Arg Glu Leu Ile Asn Ser 165 170 175 Leu Gly Ile Ile Ala Thr Asp Ala Asn Phe Ser Pro Val Leu His Cys 180 185 190 Gly Tyr Glu Val Gln Glu Val Lys Thr Ser Lys Gln Lys Leu Thr Asp 195 200 205 His Leu Thr Phe Lys Ile Ala Thr Asn Gly Ala Ile Lys Ala Val Asp 210 215 220 Ala Phe Ala Met Ala Ala Lys Ile Leu Ile Glu His Leu Asn Pro Ile 225 230 235 240 Val Ser Val Asn Glu Ser Ile Lys Asn Leu Thr Ile Ile Gln Glu Lys 245 250 255 Ala Glu Glu Arg Lys Val Lys Ser Phe Ala Lys Gln Ile Glu Glu Leu 260 265 270 Asp Phe Thr Val Arg Thr Phe Asn Cys Leu Lys Arg Ser Gly Ile His 275 280 285 Thr Leu Gln Glu Leu Leu Ser Lys Ser Leu Thr Asp Ile Arg Glu Ile 290 295 300 Arg Asn Leu Gly Lys Lys Ser Glu Arg Glu Ile Ile Lys Lys Val Gln 305 310 315 320 Glu Leu Gly Leu Lys Phe Arg Ser 325 8 329 PRT E. coli 8 Met Gln Gly Ser Val Thr Glu Phe Leu Lys Pro Arg Leu Val Asp Ile 1 5 10 15 Glu Gln Val Ser Ser Thr His Ala Lys Val Thr Leu Glu Pro Leu Glu 20 25 30 Arg Gly Phe Gly His Thr Leu Gly Asn Ala Leu Arg Arg Ile Leu Leu 35 40 45 Ser Ser Met Pro Gly Cys Ala Val Thr Glu Val Glu Ile Asp Gly Val 50 55 60 Leu His Glu Tyr Ser Thr Lys Glu Gly Val Gln Glu Asp Ile Leu Glu 65 70 75 80 Ile Leu Leu Asn Leu Lys Gly Leu Ala Val Arg Val Gln Gly Lys Asp 85 90 95 Glu Val Ile Leu Thr Leu Asn Lys Ser Gly Ile Gly Pro Val Thr Ala 100 105 110 Ala Asp Ile Thr His Asp Gly Asp Val Glu Ile Val Lys Pro Gln His 115 120 125 Val Ile Cys His Leu Thr Asp Glu Asn Ala Ser Ile Ser Met Arg Ile 130 135 140 Lys Val Gln Arg Gly Arg Gly Tyr Val Pro Ala Ser Thr Arg Ile His 145 150 155 160 Ser Glu Glu Asp Glu Arg Pro Ile Gly Arg Leu Leu Val Asp Ala Cys 165 170 175 Tyr Ser Pro Val Glu Arg Ile Ala Tyr Asn Val Glu Ala Ala Arg Val 180 185 190 Glu Gln Arg Thr Asp Leu Asp Lys Leu Val Ile Glu Met Glu Thr Asn 195 200 205 Gly Thr Ile Asp Pro Glu Glu Ala Ile Arg Arg Ala Ala Thr Ile Leu 210 215 220 Ala Glu Gln Leu Glu Ala Phe Val Asp Leu Arg Asp Val Arg Gln Pro 225 230 235 240 Glu Val Lys Glu Glu Lys Pro Glu Phe Asp Pro Ile Leu Leu Arg Pro 245 250 255 Val Asp Asp Leu Glu Leu Thr Val Arg Ser Ala Asn Cys Leu Lys Ala 260 265 270 Glu Ala Ile His Tyr Ile Gly Asp Leu Val Gln Arg Thr Glu Val Glu 275 280 285 Leu Leu Lys Thr Pro Asn Leu Gly Lys Lys Ser Leu Thr Glu Ile Lys 290 295 300 Asp Val Leu Ala Ser Arg Gly Leu Ser Leu Gly Met Arg Leu Glu Asn 305 310 315 320 Trp Pro Pro Ala Ser Ile Ala Asp Glu 325 9 329 PRT H. influenza 9 Met Gln Gly Ser Val Thr Glu Phe Leu Lys Pro Arg Leu Val Asp Ile 1 5 10 15 Glu Gln Ile Ser Ser Thr His Ala Lys Val Ile Leu Glu Pro Leu Glu 20 25 30 Arg Gly Phe Gly His Thr Leu Gly Asn Ala Leu Arg Arg Ile Leu Leu 35 40 45 Ser Ser Met Pro Gly Cys Ala Val Thr Glu Val Glu Ile Asp Gly Val 50 55 60 Leu His Glu Tyr Ser Ser Lys Glu Gly Val Gln Glu Asp Ile Leu Glu 65 70 75 80 Val Leu Leu Asn Leu Lys Gly Leu Ala Val Lys Val Gln Asn Lys Asp 85 90 95 Asp Val Ile Leu Thr Leu Asn Lys Ser Gly Ile Gly Pro Val Val Ala 100 105 110 Ala Asp Ile Thr Tyr Asp Gly Asp Val Glu Ile Val Asn Pro Asp His 115 120 125 Val Ile Cys His Leu Thr Asp Glu Asn Ala Ser Ile Ser Met Arg Ile 130 135 140 Arg Val Gln Arg Gly Arg Gly Tyr Val Pro Ala Ser Ser Arg Thr His 145 150 155 160 Thr Gln Glu Glu Arg Pro Ile Gly Arg Leu Leu Val Asp Ala Cys Tyr 165 170 175 Ser Pro Val Glu Arg Ile Ala Tyr Asn Val Glu Ala Ala Arg Val Glu 180 185 190 Gln Arg Thr Asp Leu Asp Lys Leu Val Ile Glu Leu Glu Thr Asn Gly 195 200 205 Ala Leu Glu Pro Glu Glu Ala Ile Arg Arg Ala Ala Thr Ile Leu Ala 210 215 220 Glu Gln Leu Asp Ala Phe Val Asp Leu Arg Asp Val Arg Gln Pro Glu 225 230 235 240 Ile Lys Glu Glu Lys Pro Glu Phe Xaa Asp Pro Ile Leu Leu Arg Pro 245 250 255 Val Asp Asp Leu Glu Leu Thr Val Arg Ser Ala Asn Cys Leu Lys Ala 260 265 270 Glu Thr Ile His Tyr Ile Gly Asp Leu Val Gln Arg Thr Glu Val Glu 275 280 285 Leu Leu Lys Thr Pro Asn Leu Gly Lys Lys Ser Leu Thr Glu Ile Lys 290 295 300 Asp Val Leu Ala Ser Arg Gly Leu Ser Leu Gly Met Arg Leu Glu Asn 305 310 315 320 Trp Pro Pro Ala Ser Ile Ala Glu Asp 325 10 328 PRT B. pertusis 10 Met Ser Thr Gln Gly Phe Leu Lys Pro Arg Ser Ile Glu Val Glu Pro 1 5 10 15 Val Gly Ala His His Ala Lys Ile Val Met Glu Pro Phe Glu Arg Gly 20 25 30 Tyr Gly His Thr Leu Gly Asn Ala Leu Arg Arg Ile Leu Leu Ser Ser 35 40 45 Met Thr Gly Tyr Ala Pro Thr Glu Val Gln Met Thr Gly Val Val His 50 55 60 Glu Tyr Ser Thr Ile Ala Gly Val Arg Glu Asp Val Val Asp Ile Leu 65 70 75 80 Leu Asn Leu Lys Gly Val Val Phe Lys Leu His Asn Arg Asp Glu Val 85 90 95 Thr Leu Val Leu Arg Lys Asn Gly Ala Gly Ala Val Val Ala Ser Asp 100 105 110 Ile Glu Leu Pro His Asp Val Glu Ile Ile Asn Pro Asp His Leu Ile 115 120 125 Cys Asn Leu Thr Asp Ala Gly Lys Ile Glu Met Gln Val Lys Val Glu 130 135 140 Lys Gly Arg Gly Tyr Val Pro Gly Asn Val Arg Ala Leu Ser Glu Asp 145 150 155 160 Arg Thr His Thr Ile Gly Arg Ile Val Leu Asp Ala Ser Phe Ser Pro 165 170 175 Val Arg Arg Val Ser Tyr Ala Val Glu Ser Ala Arg Val Glu Gln Arg 180 185 190 Thr Asp Leu Asp Lys Leu Val Leu Asp Ile Glu Thr Asn Gly Val Ile 195 200 205 Ser Pro Glu Glu Ala Val Arg Gln Ala Ala Arg Ile Leu Met Asp Gln 210 215 220 Ile Ser Val Phe Ala Ala Leu Glu Gly Ala Gly Asp Ala Tyr Glu Pro 225 230 235 240 Pro Val Arg Gly Thr Pro Gln Ile Asp Pro Val Leu Leu Arg Pro Val 245 250 255 Asp Asp Leu Glu Leu Thr Val Arg Ser Ala Asn Cys Leu Lys Ala Glu 260 265 270 Asn Ile Tyr Tyr Ile Gly Asp Leu Ile Gln Arg Thr Glu Asn Glu Leu 275 280 285 Leu Lys Thr Pro Asn Leu Gly Arg Lys Ser Leu Asn Glu Ile Lys Glu 290 295 300 Val Leu Ala Ala Arg Gly Leu Thr Leu Gly Met Lys Leu Glu Asn Trp 305 310 315 320 Pro Pro Leu Gly Leu Glu Arg Pro 325 11 329 PRT S. typhimurium 11 Met Gln Gly Ser Val Thr Glu Phe Leu Lys Pro Arg Leu Val Asp Ile 1 5 10 15 Glu Gln Val Ser Ser Thr His Ala Lys Val Thr Leu Glu Pro Leu Glu 20 25 30 Arg Gly Phe Gly His Thr Leu Gly Asn Ala Leu Arg Arg Ile Leu Leu 35 40 45 Ser Ser Met Pro Gly Cys Ala Val Thr Glu Val Glu Ile Asp Gly Val 50 55 60 Leu His Glu Tyr Ser Thr Lys Glu Gly Val Gln Glu Asp Ile Leu Glu 65 70 75 80 Ile Leu Leu Asn Leu Lys Gly Leu Ala Val Arg Val Gln Gly Lys Asp 85 90 95 Glu Val Ile Leu Thr Leu Asn Lys Ser Gly Ile Gly Pro Val Thr Ala 100 105 110 Ala Asp Ile Thr His Asp Gly Asp Val Glu Ile Val Lys Pro Gln His 115 120 125 Val Ile Cys His Leu Thr Asp Glu Asn Ala Ser Ile Ser Met Arg Ile 130 135 140 Lys Val Gln Arg Gly Arg Gly Tyr Val Pro Ala Ser Thr Arg Ile His 145 150 155 160 Ser Glu Glu Asp Glu Arg Pro Ile Gly Arg Leu Leu Val Asp Ala Cys 165 170 175 Tyr Ser Pro Val Glu Arg Ile Ala Tyr Asn Val Glu Ala Ala Arg Val 180 185 190 Glu Gln Arg Thr Asp Leu Asp Lys Leu Val Ile Glu Met Glu Thr Asn 195 200 205 Gly Thr Ile Asp Pro Glu Glu Ala Ile Arg Arg Ala Ala Thr Ile Leu 210 215 220 Ala Glu Gln Leu Glu Ala Phe Val Asp Leu Arg Asp Val Arg Gln Pro 225 230 235 240 Glu Val Lys Glu Glu Lys Pro Glu Phe Asp Pro Ile Leu Leu Arg Pro 245 250 255 Val Asp Asp Leu Glu Leu Thr Val Arg Ser Ala Asn Cys Leu Lys Ala 260 265 270 Glu Ala Ile His Tyr Ile Gly Asp Leu Val Gln Arg Thr Glu Val Glu 275 280 285 Leu Leu Lys Thr Pro Asn Leu Gly Lys Lys Ser Leu Thr Glu Ile Lys 290 295 300 Asp Val Leu Ala Ser Arg Gly Leu Ser Leu Gly Met Arg Leu Glu Asn 305 310 315 320 Trp Pro Pro Ala Ser Ile Ala Asp Glu 325 12 379 PRT C. trachomitis 12 Met Ser Asp Ser Ser His Asn Leu Leu Tyr Asn Lys Phe Glu Leu Pro 1 5 10 15 Glu Ser Val Lys Met Ser Pro Val Glu Gly Ala Val Gly Ser Ile Asp 20 25 30 Lys Val Ala Arg Phe Val Ala Asp Pro Leu Glu Lys Gly Met Gly His 35 40 45 Thr Leu Gly Ser Ala Leu Arg Arg Ala Leu Leu Ile Gly Leu Glu Ala 50 55 60 Pro Ala Ile Val Ser Phe Ser Met Thr Gly Val Leu His Glu Tyr Met 65 70 75 80 Ala Val Glu Gly Ile Ile Glu Asp Val Thr Asn Met Leu Leu Asn Leu 85 90 95 Lys Gly Ser Leu Leu Lys Lys Tyr Pro Leu Gln Asp Cys Glu Gly Gly 100 105 110 Arg Cys Ser Gln Lys Leu Arg Ala Thr Ile Ser Val Asp Ala Ser Asp 115 120 125 Leu Ala Ala Ala Gly Gly Gln Lys Glu Val Thr Leu Gly Asp Leu Leu 130 135 140 Gln Glu Gly Thr Phe Glu Ala Val Asn Pro Glu His Val Ile Phe Thr 145 150 155 160 Val Thr Arg Pro Met Gln Leu Glu Val Met Leu Arg Val Ala Phe Gly 165 170 175 Arg Gly Tyr Ser Pro Ser Glu Arg Ile Val Leu Glu Glu Arg Gly Met 180 185 190 Asn Glu Ile Val Leu Asp Ala Phe Ser Pro Val Val Leu Val Asn Tyr 195 200 205 Phe Val Glu Asp Thr Arg Val Gly Gln Asp Thr Asp Phe Asp Arg Leu 210 215 220 Val Leu Gln Val Glu Thr Asp Gly Arg Val Ala Pro Lys Glu Ala Val 225 230 235 240 Ala Phe Ala Thr Gln Ile Leu Ser Lys His Phe Ser Val Phe Glu Lys 245 250 255 Met Asp Glu Lys Arg Ile Val Phe Glu Glu Ala Ile Ser Val Glu Lys 260 265 270 Glu Asn Lys Asp Asp Ile Leu His Lys Leu Val Leu Gly Ile Asn Glu 275 280 285 Ile Glu Leu Ser Val Arg Leu Ile Arg Ser Thr Asn Cys Leu Ser Asn 290 295 300 Ala Asn Ile Glu Thr Ile Gly Glu Leu Val Ile Met Pro Glu Pro Arg 305 310 315 320 Leu Leu Gln Phe Arg Asn Phe Gly Lys Lys Ser Leu Cys Glu Ile Lys 325 330 335 Asn Lys Leu Lys Glu Met Lys Leu Glu Leu Gly Met Asp Leu Ser Gln 340 345 350 Phe Gly Val Gly Leu Asp Asn Val Lys Glu Lys Met Lys Trp Tyr Ala 355 360 365 Glu Lys Ile Arg Ser Ser Lys Asn Thr Lys Ala 370 375 13 24 DNA Artificial Sequence Oligonucleotide Primer 13 ctacgcaagc agggtccggg tgag 24 14 24 DNA Artificial Sequence 14 ctcacccgga ccctgcttgc gtag 24 15 64 DNA M. tuberculosis 15 agcttgcaga tctagcgatc gcagccgacg tgatacctga ccgttgttga tagtgtcggc 60 ggct 64 

What is claimed is:
 1. An isolated and purified DNA encoding M. tuberculosis RNA polymerase alpha subunit wherein said DNA has a sequence selected from the group consisting of the sequence shown in FIGS. 3A and 3B, (SEQ. ID. NO. 4) and sequence-conservative variant thereof.
 2. A DNA vector comprising the nucleic acid sequence of claim 1 operably linked to a transcription regulatory element.
 3. A cell comprising a DNA vector as defined in claim 2, wherein said cell is selected from the group consisting of bacterial, fungal, plant, insect, and mammalian cells.
 4. A cell as defined in claim 3, wherein said cell is a bacterial cell.
 5. An isolated and purified polypeptide comprising a polypeptide identified by the amino acid sequence of SEQ. ID. NO.
 5. 